Briday Mathilde, Carvalho Nicolas, Oganesyan Natalia, Chang Min-Ju, Lees Andrew, Brier Sébastien, Chenal Alexandre
Institut Pasteur, Université Paris Cité, BioNMR and HDX-MS Facility, Centre for Technological Resources and Research, Chemistry and Structural Biology Department, UMR CNRS 3528, F-75015 Paris, France.
Institut Pasteur, Université Paris Cité, Biochemistry of Macromolecular Interactions Unit, Chemistry and Structural Biology Department, UMR CNRS 3528, F-75015 Paris, France.
Int J Pharm. 2025 Apr 30;675:125535. doi: 10.1016/j.ijpharm.2025.125535. Epub 2025 Mar 30.
Carrier proteins are chemically linked to poorly immunogenic antigens to generate conjugate vaccines, significantly improving immunogenicity. CRM, a genetically detoxified diphtheria toxin (DT) mutant carrying the G52E mutation, is a widely used carrier protein as it retains lysine residues for antigen conjugation. In the past, CRM has been expressed in Corynebacterium diphtheriae, but low yields and high costs have prompted the exploration of alternative expression systems. Although high-yield expression and native refolding of CRM in E. coli are challenging due to its reducing cytoplasm, recent advances have enabled the production of soluble and well-folded recombinant CRM proteins, namely EcoCRM® and EcoCRM®. In this study, we use Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) to compare the structural dynamics of EcoCRM and EcoCRM with DT wild-type. Our HDX-MS data show that the presence or the absence of the N-terminal methionine does not affect the structural dynamics of the two recombinant EcoCRM proteins. Furthermore, our results elucidate the molecular mechanism underlying the lack of toxicity of CRM compared to DT wild-type: the G52E mutation in the CRM proteins exclusively alters the stability of the NAD-binding pocket and induces allosteric effects within the receptor-binding domain. Altogether, these insights support the substitution of CRM produced by C. diphtheriae with the recombinant EcoCRM and EcoCRM proteins produced in E. coli, offering a cost-effective solution for use in conjugate vaccines. Data are available via ProteomeXchange with identifier PXD057388.
载体蛋白与免疫原性较差的抗原进行化学连接以生成结合疫苗，显著提高免疫原性。CRM是一种携带G52E突变的基因解毒白喉毒素（DT）突变体，是一种广泛使用的载体蛋白，因为它保留了用于抗原偶联的赖氨酸残基。过去，CRM已在白喉棒状杆菌中表达，但产量低和成本高促使人们探索替代表达系统。尽管由于大肠杆菌的细胞质具有还原性，在其中高产表达和天然重折叠CRM具有挑战性，但最近的进展使得能够生产可溶性且折叠良好的重组CRM蛋白，即EcoCRM®和EcoCRM®。在本研究中，我们使用氢/氘交换质谱（HDX-MS）来比较EcoCRM和EcoCRM与DT野生型的结构动力学。我们的HDX-MS数据表明，N端甲硫氨酸的存在与否不影响这两种重组EcoCRM蛋白的结构动力学。此外，我们的结果阐明了与DT野生型相比CRM缺乏毒性的分子机制：CRM蛋白中的G52E突变仅改变了NAD结合口袋的稳定性，并在受体结合域内诱导了变构效应。总之，这些见解支持用大肠杆菌中产生的重组EcoCRM和EcoCRM蛋白替代白喉棒状杆菌产生的CRM，为结合疫苗的使用提供了一种经济高效的解决方案。数据可通过ProteomeXchange获得，标识符为PXD057388。
